Antisense modulation of MDMX expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of MDMX. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding MDMX. Methods of using these compounds for modulation of MDMX expression and for treatment of diseases associated with expression of MDMX are provided.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulating the expression of MDMX. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding human MDMX. Such oligonucleotides have been shown to modulate the expression of MDMX.

BACKGROUND OF THE INVENTION

Carcinogenesis is the result of the deregulation of the fundamental biological processes involved in cell growth and differentiation. Two proteins, p53 and MDM2, appear to play reciprocal roles in regulating the cellular progression toward tumorigenicity.

p53, a tumor suppressor gene, acts as a transcription factor and negatively regulates cell growth by controlling entry into the cell cycle. Mutations in this gene are found in over 60% of all cancers. p53 acts to protect the cell after DNA damage by activating the transcription of target genes which initiate cell cycle arrest until the damage can be repaired. Alterations in the sequence, expression, subcellular localization, or concentration of the p53 protein product can result in the tumor phenotype (Momand and Zambetti, J. Cell. Biochem., 1997, 64, 343-352).

In contrast to the cell survival properties of p53, a protein called MDM2, acts in the opposite manner producing an oncogenic phenotype when overexpressed. MDM2 promotes cellular growth by directly binding to p53 thereby sequestering the protein and thus inactivating its tumor suppressor function (Momand and Zambetti, J. Cell. Biochem., 1997, 64, 343-352).

Recently, another p53-associating protein was isolated in mice and based on its structural similarity to MDM2 was called MDMX (Shvarts et al., Embo J., 1996, 15, 5349-5357). The human homolog has been reported and shares the same p53-associated features as the murine protein (Shvarts et al., Genomics, 1997, 43, 34-42).

MDMX (also known as mdm2-like p53 binding protein) was shown to bind to p53 in vivo and inhibit the trascription activation function of wild type p53. However, unlike MDM2, MDMX is not regulated in a p53-dependent manner. The MDMX transcript was found to be expressed in all human tissues with the highest levels found in the thymus and the lowest in the colon. By fluorescence in situ hybridization (FISH) analysis, MDMX was localized to chromosome 1q32, a region of the genome found to be amplified in several liposarcomas, possibly implicating MDMX in carcinogenesis (Shvarts et al., Genomics, 1997, 43, 34-42).

The nucleic acid sequence of the mouse MDMX gene is disclosed in WO96/41875 (Van Der Eb et al., 1996, ). Also disclosed in WO96/41875 is a vector containing the DNA, transformants containing this vector and methods of producing MDMX with this vector. In addition, this PCT application discloses methods of screening for substances which may bind p53 using the MDMX vector in hybridization assays.

Currently, there are no known therapeutic agents which effectively inhibit the synthesis of MDMX. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting MDMX function.

It is anticipated that antisense oligonucleotides against MDMX may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the effective and specific modulation of MDMX expression.

SUMMARY OF THE INVENTION

The present invention is directed to antisense compounds, particularly oligonucleotides, which are targeted to a nucleic acid encoding MDMX, and which modulate the expression of MDMX. Pharmaceutical and other compositions comprising the antisense compounds of the invention are also provided. Further provided are methods of modulating the expression of MDMX in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of MDMX by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligomeric antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding MDMX, ultimately modulating the amount of MDMX produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding MDMX. As used herein, the terms "target nucleic acid" and "nucleic acid encoding MDMX" encompass DNA encoding MDMX, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as "antisense". The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of MDMX. In the context of the present invention, "modulation" means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.

It is preferred to target specific nucleic acids for antisense. "Targeting" an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding MDMX. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon," the "start codon" or the "AUG start codon". A minority of genes have a translation initiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the terms "translation initiation codon" and "start codon" can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, "start codon" and "translation initiation codon" refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding MDMX, regardless of the sequence(s) of such codons.

It is also known in the art that a translation termination codon (or "stop codon") of a gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA, respectively). The terms "start codon region" and "translation initiation codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon. Similarly, the terms "stop codon region" and "translation termination codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon.

The open reading frame (ORF) or "coding region," which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3' untranslated region (3'UTR), known in the art to refer to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA or corresponding nucleotides on the gene. The 5' cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5' cap region may also be a preferred target region.

Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as "introns," which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as "exons" and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.

Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

In the context of this invention, "hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. "Complementary," as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.

Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use.

The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotides have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans. In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 30 nucleobases. Particularly preferred are antisense oligonucleotides comprising from about 8 to about 30 nucleobases (i.e. from about 8 to about 30 linked nucleosides). As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thiono-alkylphosphonates, thionoalkylphosphotriesters, and borano-phosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S.Pat. No.: 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and U.S. Pat. No. 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. : 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and U.S. Pat. No. 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. : 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular --CH₂ --NH--O--CH₂ --, --CH₂ --N(CH₃)--O--CH₂ --[known as a methylene (methylimino) or MMI backbone], --CH₂ --O--N(CH₃)--CH₂ --, --CH₂ --N(CH₃)--N(CH₃) --CH₂ -- and --O--N(CH₃)--CH₂ --CH₂ --[wherein the native phosphodiester backbone is represented as --O--P--O--CH₂ --] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2' position: OH; F; O--, S--, or N-alkyl; O--, S--, or N-alkenyl; O--, S-- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n) O]_(m) CH₃, O(CH₂)_(n) OCH₃, O(CH₂)_(n) NH₂, O(CH₂)_(n) CH₃, O(CH₂)_(n) NH₂, and O(CH₂)_(n) ON[(CH₂)_(n) CH₃) ]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy (2'--O--CH₂ CH₂ OCH₃, also known as 2'--O--(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ ON(CH₃)₂ group, also known as 2'-DMAOE, as described in examples hereinbelow.

Other preferred modifications include 2'-methoxy (2'--O--CH₃), 2'-aminopropoxy (2'--OCH₂ CH₂ CH₂ NH₂) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. : 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. : 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937.

Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and U.S. Pat. No. 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. "Chimeric" antisense compounds or "chimeras," in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 35 5,652,355; 5,652,356; and U.S. Pat. No. 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

The antisense compounds of the invention are synthesized in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules. The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include, but are not limited to, U.S. Pat. Nos. : 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and U.S. Pat. No. 5,595,756, each of which is herein incorporated by reference.

The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

The term "prodrug" indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 to Imbach et al.

The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., "Pharmaceutical Salts," J. of Pharma Sci., 1977, 66, 1-19). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a "pharmaceutical addition salt" includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid. Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of MDMX is treated by administering antisense compounds in accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example.

The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding MDMX, enabling sandwich and other assays to easily be constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding MDMX can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of MDMX in a sample may also be prepared.

The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2'--O-methoxyethyl modification are believed to be particularly useful for oral administration.

Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.

Emulsions

The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising of two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be either water-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of reasons of ease of formulation, efficacy from an absorption and bioavailability standpoint. (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

In one embodiment of the present invention, the compositions of oligonucleotides and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (S0750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories--surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes. As the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).

Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C₁₂ 15G, that contains a PEG moiety. Ilium et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising antisense oligonucleotides targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

Surfactants: In connection with the present invention, surfactants (or "surface-active agents") are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term "bile salts" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such As polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides.

Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

Excipients

In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Other Components

The compositions of the present invention may additionally contain other adjunct components conventionally 20 found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include, but are not limited to, anticancer drugs such as daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 1206-1228). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀ s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1

Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2'-alkoxy amidites

2'-Deoxy and 2'-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham MA or Glen Research, Inc. Sterling Va.). Other 2'-O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Pat. No. 5,506,351, herein incorporated by reference. For oligonucleotides synthesized using 2'-alkoxy amidites, the standard cycle for unmodified oligonucleotides was utilized, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds.

Oligonucleotides containing 5-methyl-2'-deoxycytidine (5-Me-C) nucleotides were synthesized according to published methods [Sanghvi, et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.).

2'-Fluoro amidites

2'-Fluorodeoxyadenosine amidites

2'-fluoro oligonucleotides were synthesized as described previously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No. 5,670,633, herein incorporated by reference. Briefly, the protected nucleoside N6-benzoyl-2'-deoxy-2'-fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and by modifying literature procedures whereby the 2'-alpha-fluoro atom is introduced by a S_(n) 2-displacement of a 2'-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3', 5'-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N6-benzoyl groups was accomplished using standard methodologies and standard methods were used to obtain the 5'-dimethoxytrityl-(DMT) and 5'-DMT-3'-phosphoramidite intermediates.

2'-Fluorodeoxyguanosine

The synthesis of 2'-deoxy-2'-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-beta-D-arabinofuranosylguanine as starting material, and conversion to the intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give diisobutyryl di-THP protected arabinofuranosylguanine. Selective O-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies were used to obtain the 5'-DMT- and 5'-DMT-3'-phosphoramidites.

2'-Fluorouridine

Synthesis of 2'-deoxy-2'-fluorouridine was accomplished by the modification of a literature procedure in which 2,2'-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine. Standard procedures were used to obtain the 5'-DMT and 5'-DMT-3'phosphoramidites.

2'-Fluorodeoxycytidine

2'-deoxy-2'-fluorocytidine was synthesized via amination of 2'-deoxy-2'-fluorouridine, followed by selective protection to give N4-benzoyl-2'-deoxy-2'-fluorocytidine. Standard procedures were used to obtain the 5'-DMT and 5'-DMT-3'phosphoramidites.

2'-O-(2-Methoxyethyl) modified amidites

2'-O-Methoxyethyl-substituted nucleoside amidites are prepared as follows, or alternatively, as per the methods of Martin, P., Helvetica Chimica Acta, 1995, 78, 486-504.

2,2'-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released in a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure. The resulting syrup was poured into diethylether (2.5 L), with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL). The solution was poured into fresh ether (2.5 L) to yield a stiff gum. The ether was decanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for 24 h) to give a solid that was crushed to a light tan powder (57 g, 85% crude yield). The NMR spectrum was consistent with the structure, contaminated with phenol as its sodium salt (ca. 5%). The material was used as is for further reactions (or it can be purified further by column chromatography using a gradient of methanol in ethyl acetate (10-25%) to give a white solid, mp 222-4° C.).

2'-O-Methoxyethyl-5-methyluridine

2,2'-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 L stainless steel pressure vessel and placed in a pre-heated oil bath at 160° C. After heating for 48 hours at 155-160° C., the vessel was opened and the solution evaporated to dryness and triturated with MeOH (200 mL). The residue was suspended in hot acetone (1 L). The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate evaporated. The residue (280 g) was dissolved in CH₃ CN (600 mL) and evaporated. A silica gel column (3 kg) was packed in CH₂ Cl₂ /acetone/MeOH (20:5:3) containing 0.5% Et₃ NH. The residue was dissolved in CH₂ Cl₂ (250 mL) and adsorbed onto silica (150 g) prior to loading onto the column. The product was eluted with the packing solvent to give 160 g (63%) of product. Additional material was obtained by reworking impure fractions.

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine

2'-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporated with pyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the mixture stirred at room temperature for one hour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the reaction stirred for an additional one hour. Methanol (170 mL) was then added to stop the reaction. HPLC showed the presence of approximately 70% product. The solvent was evaporated and triturated with CH₃ CN (200 mL) The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phase was dried over Na₂ SO₄, filtered and evaporated. 275 g of residue was obtained. The residue was purified on a 3.5 kg silica gel column, packed and eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5% Et₃ NH. The pure fractions were evaporated to give 164 g of product. Approximately 20 g additional was obtained from the impure fractions to give a total yield of 183 g (57%).

3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room temperature for 24 hours. The reaction was monitored by TLC by first quenching the TLC sample with the addition of MeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL) was added and the mixture evaporated at 35° C. The residue was dissolved in CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodium bicarbonate and 2×200 mL of saturated NaCl. The water layers were back extracted with 200 mL of CHCl₃. The combined organics were dried with sodium sulfate and evaporated to give 122 g of residue (approx. 90% product). The residue was purified on a 3.5 kg silica gel column and eluted using EtOAc/hexane(4:1). Pure product fractions were evaporated to yield 96 g (84%). An additional 1.5 g was recovered from later fractions.

3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving 3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in CH₃ CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH₃ CN (1 L), cooled to -5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃ was added dropwise, over a 30 minute period, to the stirred solution maintained at 0-10° C., and the resulting mixture stirred for an additional 2 hours. The first solution was added dropwise, over a 45 minute period, to the latter solution. The resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound.

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine

A solution of 3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH₄ OH (30 mL) was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2×200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. MeOH (400 mL) saturated with NH₃ gas was added and the vessel heated to 100° C. for 2 hours (TLC showed complete conversion). The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL). The organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound.

N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine

2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, TLC showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MeOH (200 mL). The residue was dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give a residue (96 g). The residue was chromatographed on a 1.5 kg silica column using EtOAc/hexane (1:1) containing 0.5% Et₃ NH as the eluting solvent. The pure product fractions were evaporated to give 90 g (90%) of the title compound.

N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine-3'-amidite

N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) was dissolved in CH₂ Cl₂ (1 L). Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere. The resulting mixture was stirred for 20 hours at room temperature (TLC showed the reaction to be 95% complete). The reaction mixture was extracted with saturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes were back-extracted with CH₂ Cl₂ (300 mL), and the extracts were combined, dried over MgSO₄ and concentrated. The residue obtained was chromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g (87%) of the title compound.

2'-O-(Aminooxyethyl) nucleoside amidites and 2'-O-(dimethylaminooxyethyl) nucleoside amidites

2'-(Dimethylaminooxyethoxy) nucleoside amidites

2'-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the art as 2'-O-(dimethylaminooxyethyl) nucleoside amidites] are prepared as described in the following paragraphs. Adenosine, cytidine and guanosine nucleoside amidites are prepared similarly to the thymidine (5-methyluridine) except the exocyclic amines are protected with a benzoyl moiety in the case of adenosine and cytidine and with isobutyryl in the case of guanosine.

5'-O-tert-Butyldiphenylsilyl -O² -2'-anhydro-5-methyluridine

O² -2'-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. The reaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L) and brine (1 L). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) and the solution was cooled to -10° C. The resulting crystalline product was collected by filtration, washed with ethyl ether (3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMR were consistent with pure product.

5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine

In a 2 L stainless steel, unstirred pressure reactor was added borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and with manual stirring, ethylene glycol (350 mL, excess) was added cautiously at first until the evolution of hydrogen gas subsided. 5'-O-tert-Butyldiphenylsilyl-O² -2'-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manual stirring. The reactor was sealed and heated in an oil bath until an internal temperature of 160° C. was reached and then maintained for 16 h (pressure <100 psig). The reaction vessel was cooled to ambient and opened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T side product, ethyl acetate) indicated about 70% conversion to the product. In order to avoid additional side product formation, the reaction was stopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath (40-100° C.) with the more extreme conditions used to remove the ethylene glycol. [Alternatively, once the low boiling solvent is gone, the remaining solution can be partitioned between ethyl acetate and water. The product will be in the organic phase.] The residue was purified by column chromatography (2 kg silica gel, ethyl acetate-hexanes gradient 1:1 to 4:1). The appropriate fractions were combined, stripped and dried to product as a white crisp foam (84 g, 50%), contaminated starting material (17.4 g) and pure reusable starting material 20 g. The yield based on starting material less pure recovered starting material was 58%. TLC and NMR were consistent with 99% pure product.

2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine

5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine (20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried over P₂ O₅ under high vacuum for two days at 40° C. The reaction mixture was flushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) was added to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the reaction mixture. The rate of addition is maintained such that resulting deep red coloration is just discharged before adding the next drop. After the addition was complete, the reaction was stirred for 4 hrs. By that time TLC showed the completion of the reaction (ethylacetate:hexane, 60:40). The solvent was evaporated in vacuum. Residue obtained was placed on a flash column and eluted with ethyl acetate:hexane (60:40), to get 2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine as white foam (21.819 g, 86%).

5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine (3.1 g, 4.5 mmol) was dissolved in dry CH₂ Cl₂ (4.5 mL) and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at -10° C. to 0° C. After 1 h the mixture was filtered, the filtrate was washed with ice cold CH₂ Cl₂ and the combined organic phase was washed with water, brine and dried over anhydrous Na₂ SO₄. The solution was concentrated to get 2'-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5 mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was strirred for 1 h. Solvent was removed under vacuum; residue chromatographed to get 5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy) ethyl]-5-methyluridine as white foam (1.95 g, 78%).

5'-O-tert-Butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added to this solution at 10° C. under inert atmosphere. The reaction mixture was stirred for 10 minutes at 10° C. After that the reaction vessel was removed from the ice bath and stirred at room temperature for 2 h, the reaction monitored by TLC (5% MeOH in CH₂ Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extracted with ethyl acetate (2×20 mL). Ethyl acetate phase was dried over anhydrous Na₂ SO₄, evaporated to dryness. Residue was dissolved in a solution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) was added and the reaction mixture was stirred at room temperature for 10 minutes. Reaction mixture cooled to 10° C. in an ice bath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reaction mixture stirred at 10° C. for 10 minutes. After 10 minutes, the reaction mixture was removed from the ice bath and stirred at room temperature for 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was added and extracted with ethyl acetate (2×25 mL). Ethyl acetate layer was dried over anhydrous Na₂ SO₄ and evaporated to dryness . The residue obtained was purified by flash column chromatography and eluted with 5% MeOH in CH₂ Cl₂ to get 5'-O-tert-butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%).

2'-O-(dimethylaminooxyethyl)-5-methyluridine

Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). This mixture of triethylamine-2HF was then added to 5'-O-tert-butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reaction was monitored by TLC (5% MeOH in CH₂ Cl₂). Solvent was removed under vacuum and the residue placed on a flash column and eluted with 10% MeOH in CH₂ Cl₂ to get 2'-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%).

5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine 2'-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) was dried over P₂ O₅ under high vacuum overnight at 40° C. It was then co-evaporated with anhydrous pyridine (20 mL). The residue obtained was dissolved in pyridine (11 mL) under argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4'-dimethoxytrityl chloride (880 mg, 2.60 mmol) was added to the mixture and the reaction mixture was stirred at room temperature until all of the starting material disappeared. Pyridine was removed under vacuum and the residue chromatographed and eluted with 10% MeOH in CH₂ Cl₂ (containing a few drops of pyridine) to get 5'-O-DMT-2'-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL). To the residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and dried over P₂ O₅ under high vacuum overnight at 40° C. Then the reaction mixture was dissolved in anhydrous acetonitrile (8.4 mL) and 2-cyanoethyl-N,N,N¹,N¹ -tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at ambient temperature for 4 hrs under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated, then the residue was dissolved in ethyl acetate (70 mL) and washed with 5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrous Na₂ SO₄ and concentrated. Residue obtained was chromatographed (ethyl acetate as eluent) to get 5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%).

2'-(Aminooxyethoxy) nucleoside amidites

2'-(Aminooxyethoxy) nucleoside amidites [also known in the art as 2'-O-(aminooxyethyl) nucleoside amidites] are prepared as described in the following paragraphs. Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramiditel].

The 2'-O-aminooxyethyl guanosine analog may be obtained by selective 2'-O-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2'-O-(2-ethylacetyl) diaminopurine riboside along with a minor amount of the 3'-O-isomer. 2'-O-(2-ethylacetyl) diaminopurine riboside may be resolved and converted to 2'-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection procedures should afford 2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

Example 2

Oligonucleotide Synthesis

Unsubstituted and substituted phosphodiester (P=O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P=S) are synthesized as for the phosphodiester oligonucleotides except the standard oxidation bottle was replaced by 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation wait step was increased to 68 sec and was followed by the capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (18 h), the oligonucleotides were purified by precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl solution.

Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

3'-Deoxy-3'-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No., 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

3'-Deoxy-3'-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3

Oligonucleoside Synthesis

Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethyl-hydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligo-nucleosides, also identified as amide-4 linked oligonucleo-sides, as well as mixed backbone compounds having, for instance, alternating MMI and P=O or P=S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

PNA Synthesis

Peptide nucleic acids (PNAs) are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference.

Example 5

Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the "gap" segment of linked nucleosides is positioned between 5' and 3' "wing" segments of linked nucleosides and a second "open end" type wherein the "gap" segment is located at either the 3' or the 5' terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as "gapmers" or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as "hemimers" or "wingmers".

[2'-O-Me]--[2'-deoxy]--[2'-O-Me] Chimeric Phosphorothioate Oligonucleotides

Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate and 2'-deoxy phosphorothioate oligo-nucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 380B, as above. Oligonucleotides are synthesized using the automated synthesizer and 2'-deoxy-5'-dimethoxytrityl-3'-O-phosphor-amidite for the DNA portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for 5' and 3' wings. The standard synthesis cycle is modified by increasing the wait step after the delivery of tetrazole and base to 600 s repeated four times for RNA and twice for 2'-O-methyl. The fully protected oligonucleotide is cleaved from the support and the phosphate group is deprotected in 3:1 ammonia/ethanol at room temperature overnight then lyophilized to dryness. Treatment in methanolic ammonia for 24 hrs at room temperature is then done to deprotect all bases and sample was again lyophilized to dryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at room temperature to deprotect the 2' positions. The reaction is then quenched with 1M TEAA and the sample is then reduced to 1/2 volume by rotovac before being desalted on a G25 size exclusion column. The oligo recovered is then analyzed spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[2'-O-(2-Methoxyethyl)]--[2'-deoxyl]--[2'-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[2'-O-(2-methoxyethyl)]--[2'-deoxy]--[-2'-O-(methoxy-ethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2'-O-methyl chimeric oligonucleotide, with the substitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites.

[2'-O-(2-Methoxyethyl)Phosphodiester]--[2'-deoxy Phosphorothioate]--[2'-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides

[2'-O-(2-methoxyethyl phosphodiester]--[2'-deoxy phos-phorothioate]--[2'-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2'-O-methyl chimeric oligonucleotide with the substitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites, oxidization with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3, H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

Oligonucleotide Isolation

After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55° C. for 18 hours, the oligonucleotides or oligonucleosides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on denaturing gels and judged to be at least 85% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in synthesis were periodically checked by BP nuclear magnetic resonance spectroscopy, and for some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7

Oligonucleotide Synthesis--96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a standard 96 well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3, H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per known literature or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected with concentrated NH₄ OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8

Oligonucleotide Analysis--96 Well Plate Format

The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9

Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following four cell types are provided for illustrative purposes, but other cell types can be routinely used.

T-24 cells:

The transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

A549 cells:

The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

NHDF cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

HEK cells:

Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

Treatment with Antisense Compounds:

When cells reached 80% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Gibco BRL) and the desired oligonucleotide at a final concentration of 150 nM. After 4 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16 hours after oligonucleotide treatment.

Example 10

Analysis of Oligonucleotide Inhibition of MDMX Expression

Antisense modulation of MDMX expression can be assayed in a variety of ways known in the art. For example, MDMX mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F.M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions. Other methods of PCR are also known in the art.

MDMX protein levels can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to MDMX can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

Example 11

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., Clin. Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C. was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Example 12

Total RNA Isolation

Total mRNA was isolated using an RNEASY ₉₆ ™ kit and buffers purchased from Qiagen Inc. (Valencia Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 100 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY ₉₆ ™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL of Buffer RWl was added to each well of the RNEASY ₉₆ ™ plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was then added to each well of the RNEASY ₉₆ ™ plate and the vacuum applied for a period of 15 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 60 μL water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 μL water.

Example 13

Real-time Quantitative PCR Analysis of MDMX mRNA Levels

Quantitation of MDMX mRNA levels was determined by real-time quantitative PCR using the ABI PRISM™ 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR, in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., JOE or FAM, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 5' end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 3' end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3' quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5'-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular (six-second) intervals by laser optics built into the ABI PRISM™ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

PCR reagents were obtained from PE-Applied Biosystems, Foster City, Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail (1×TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of DATP, dCTP and dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5 Units MuLV reverse transcriptase) to 96 well plates containing 25 μL poly(A) mRNA solution. The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension). MDMX probes and primers were designed to hybridize to the human MDMX sequence, using published sequence information (GenBank accession number AF007111, incorporated herein as SEQ ID NO:1).

For MDMX the PCR primers were:

forward primer: TGCCGTAAGTTTTACCAACAGACT (SEQ ID NO: 2)

reverse primer: GGCAGTTTTGGTCGTACCTGAT (SEQ ID NO: 3) and the PCR probe was: FAM-TCATTTTCCACCTCTGCTCAGTGTTCAACATC-TAMRA (SEQ ID NO: 4) where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

For GAPDH the PCR primers were:

forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 5)

reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 6)and the PCR probe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC- TAMRA 3' (SEQ ID NO: 7) where JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.

Example 14

Northern Blot Analysis of MDMX mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST "B" Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio.). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST "B" Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ Uv Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.).

Membranes were probed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions with a MDMX specific probe prepared by PCR using the forward primer TGCCGTAAGTTTTACCAACAGACT (SEQ ID NO: 2) and the reverse primer GGCAGTTTTGGTCGTACCTGAT (SEQ ID NO: 3). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.). Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15

Antisense Inhibition of MDMX Expression-Phosphorothioate 2'-MOE Gapmer Oligonucleotides

In accordance with the present invention, a series of oligonucleotides targeted to human MDMX were synthesized. The oligonucleotide sequences are shown in Table 1. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. AF007111), to which the oligonucleotide binds.

All compounds in Table 1 are chimeric oligonucleotides ("gapmers") 20 nucleotides in length, composed of a central "gap" region consisting of ten 2'-deoxynucleotides, which is flanked on both sides (5' and 3' directions) by five-nucleotide "wings". The wings are composed of 2'-methoxyethyl (2'-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P=S) throughout the oligonucleotide. Cytidine residues in the 2'-MOE wings are 5-methylcytidines.

Data were obtained by real-time quantitative PCR as described in other examples herein and are averaged from two experiments. If present, "N.D." indicates "not done".

                                      TABLE 1                                      __________________________________________________________________________     Inhibition of MDMX mRNA levels by chimeric phosphorothioate                     oligonucleotides having 2'-MOE wings and a deoxy gap                                     TARGET           %     SEQ ID                                                                          ISIS# REGION SITE SEQUENCE Inhibition                                         NO.                                          __________________________________________________________________________     30988                                                                               5' UTR                                                                               55    cagctccatgagtcgctccc                                                                       0    8                                              31147 5' UTR 58 cggcagctccatgagtcgct N.D. 9                                    30989 5' UTR 79 gcagtctgttggtaaaactt 83 10                                     31148 5' UTR 81 ctgcagtctgttggtaaaac N.D. 11                                   30990 5' UTR 84 aaactgcagtctgttggtaa 75 12                                     31149 5' UTR 87 aagaaactgcagtctgttgg N.D. 13                                   30991 Start 98 ttttggtagtgaagaaactg 84 14                                       Codon                                                                         30992 Start 99 attttggtagtgaagaaact 75 15                                       Codon                                                                         30993 Start 100 cattttggtagtgaagaaac 80 16                                      Codon                                                                         30994 Start 101 tcattttggtagtgaagaaa 85 17                                      Codon                                                                         30995 Start 102 gtcattttggtagtgaagaa 88 18                                      Codon                                                                         30996 Start 103 tgtcattttggtagtgaaga 79 19                                      Codon                                                                         30997 Start 104 atgtcattttggtagtgaag 84 20                                      Codon                                                                         30998 Start 105 gatgtcattttggtagtgaa  9 21                                      Codon                                                                         30999 Start 106 tgatgtcattttggtagtga 78 22                                      Codon                                                                         31000 Start 107 atgatgtcattttggtagtg 74 23                                      Codon                                                                         31001 Start 108 aatgatgtcattttggtagt 87 24                                      Codon                                                                         31002 Start 118 agaggtggaaaatgatgtca 84 25                                      Codon                                                                         31003 Start 119 cagaggtggaaaatgatgtc 87 26                                      Codon                                                                         31150 Coding 123 tgagcagaggtggaaaatga N.D. 27                                  31151 Coding 145 agcactgtcagatgttgaac N.D. 28                                  31004 Coding 147 caagcactgtcagatgttga 91 29                                    31005 Coding 154 gatcctgcaagcactgtcag 78 30                                    31152 Coding 173 gattgatttgtccaggagag N.D. 31                                  31006 Coding 189 agttttggtcgtacctgatt 78 32                                    31153 Coding 209 gcaaatcttcaaaagcggc N.D. 33                                   31007 Coding 212 catgcaaaatcttcaaaagc 70 34                                    31154 Coding 217 tgctgcatgcaaaatcttca N.D. 35                                  31008 Coding 249 tctttaacagtgaacatttc 70 36                                    31155 Coding 253 gacctctttaacagtgaaca N.D. 37                                  31009 Coding 263 aatagtgcatgacctcttta 84 38                                    31156 Coding 275 tgtactgacctaaatagtgc N.D. 39                                  31010 Coding 284 tcaccattatgtactgacct 90 40                                    31011 Coding 298 atcataaagttgcttcacca 85 41                                    31157 Coding 303 tgctgatcataaagttgctt N.D. 42                                  31012 Coding 320 ataccatatgctgctcctgc 88 43                                    31158 Coding 323 aatataccatatgctgctcc N.D. 44                                  31013 Coding 352 cagtagttctcccaaaagat 88 45                                    31159 Coding 360 tgacgtcccagtagttctcc N.D. 46                                  31014 Coding 365 agctctgacgtcccagtagt 74 47                                    31160 Coding 388 agggcttgggttctttacgg N.D. 48                                  31015 Coding 402 agcatatcatagagagggct 82 49                                    31161 Coding 407 ttcttagcatatcatagaga N.D. 50                                  31016 Coding 429 gtggctaaagtgacaagatt 89 51                                    31162 Coding 443 catctgtagtagcagtggct N.D. 52                                  31017 Coding 451 ctgagcagcatctgtagtag 84 53                                    31163 Coding 460 agcgagagtctgagcagcat N.D. 54                                  31018 Coding 476 tgtgatcctgtgcgagagcg 76 55                                    31164 Coding 482 ccatactgtgatcctgtgcg N.D. 56                                  31019 Coding 505 cagttggtcttgacttggaa 71 57                                    31165 Coding 512 tttgcttcagttggtcttga N.D. 58                                  31020 Coding 519 tctgcactttgcttcagttg 69 59                                    31166 Coding 525 ctttcctctgcactttgctt N.D. 60                                  31167 Coding 584 gctctgaggtaggcagtgtg N.D. 61                                  31021 Coding 594 atgcatttatgctctgaggt  0 62                                    31168 Coding 623 caattaagtcttcatcttct N.D. 63                                  31022 Coding 661 aaggtccagcctagatgttt 89 64                                    31169 Coding 672 tcctcaaatccaaggtccag N.D. 65                                  31023 Coding 681 acatcccactcctcaaatcc 70 66                                    31170 Coding 686 cagctacatcccactcctca N.D. 67                                  31024 Coding 695 aaggcaggccagctacatcc 78 68                                    31171 Coding 724 gttgcttctcaagtttccta N.D. 69                                  31172 Coding 753 tcagttgagccattacttct N.D. 70                                  31035 Coding 762 gtctgtaaatcagttgagcc 78 71                                    31173 Coding 776 ccacatcctgatttgtctgt N.D. 72                                  31036 Coding 788 caatggcagtacccacatcc 87 73                                    31027 Coding 805 atctgtagtatctgaaacaa 71 74                                    31174 Coding 814 ccacaagtcatctgtagtat N.D. 75                                  31028 Coding 851 caacacctaactgctctgat 87 76                                    31175 Coding 859 ttttattccaacacctaact N.D. 77                                  31029 Coding 875 tatcagcagcttcaactttt 79 78                                    31030 Coding 890 cacttgtttgttcagtatca 78 79                                    31176 Coding 901 ccctacttcttcacttgttt N.D. 80                                  31031 Coding 956 agtcctccaggtcatcattt 83 81                                    31177 Coding 986 ctacatcggtatcatcactt N.D. 82                                  31178 Coding 992 taacctctacatcggtatca N.D. 83                                  31032 Coding 996 gaggtaacctctacatcggt 75 84                                    31179 Coding 1018 agtacactgccactcatcct N.D. 85                                 31033 Coding 1034 taaatttcttgcattcagta 87 86                                   31180 Coding 1075 caaggcccaacaacgaaaac N.D. 87                                 31181 Coding 1080 ttcctcaaggcccaacaacg N.D. 88                                 31034 Coding 1096 atctgaataccaatccttcc 85 89                                   31182 Coding 1103 ttgaacaatctgaataccaa N.D. 90                                 31035 Coding 1110 gttaactttgaacaatctga 81 91                                   31183 Coding 1117 agaatgggttaactttgaac N.D. 92                                 31036 Coding 1133 tatcagacgtggagagagaa 87 93                                   31184 Coding 1152 ttttcaggtatggcagtgat N.D. 94                                 31037 Coding 1160 cattttccttttcaggtatg 83 95                                   31185 Coding 1168 atttccttcattttcctttt N.D. 96                                 31038 Coding 1173 acatcatttccttcattttc  0 97                                   31186 Coding 1180 atcagggacatcatttcctt N.D. 98                                 31039 Coding 1189 tcttcgacaatcagggacat 91 99                                   31187 Coding 1195 aatggttcttcgacaatcag N.D. 100                                31040 Coding 1210 aacgacaggagccgaaatgg 79 101                                  31188 Coding 1218 ttaggtctaacgacaggagc N.D. 102                                31041 Coding 1231 tatatacgcatctttaggtc 92 103                                  31189 Coding 1236 ttctttatatacgcatcttt N.D. 104                                31042 Coding 1282 caagaattccactgagttgc 87 105                                  31190 Coding 1296 ctgtgagccaaatccaagaa N.D. 106                                31043 Coding 1309 ttggctttcagaactgtgag  0 107                                  31191 Coding 1317 atggtctcttggctttcaga N.D. 108                                31044 Coding 1337 actgttctcccatgcttgag 80 109                                  31192 Coding 1347 aggttatctaactgttctcc N.D. 110                                31193 Coding 1358 tctgttcagaaaggttatct N.D. 111                                31045 Coding 1372 ttctgtatctgttctctgtt 88 112                                  31194 Coding 1381 ctccatgttttctgtatctg N.D. 113                                31046 Coding 1386 caatcctccatgttttctgt 89 114                                  31195 Coding 1407 catggcttcaagagattctg N.D. 115                                31047 Coding 1432 tcgtggtcttttctcacata 90 116                                  31196 Coding 1438 cccgtctcgtggtcttttct N.D. 117                                31048 Coding 1447 aataatgttcccgtctcgtg 85 118                                  31197 Coding 1456 ccttccatgaataatgttcc N.D. 119                                31198 Coding 1471 gacaagatggcccgtccttc N.D. 120                                31049 Coding 1484 agtgaaaacaagtgacaaga 83 121                                  31050 Coding 1493 ttctggcacagtgaaaacaa 90 122                                  31199 Coding 1503 ttctttagtcttctggcaca N.D. 123                                31200 Coding 1536 tctttcttgcaaatagggca N.D. 124                                31051 Coding 1554 ttaataaccagctgaatctc 84 125                                  31201 Stop 1582 ttcgtactaccattatgcta N.D. 127                                   Codon                                                                         31202 Stop 1583 gttcgtactaccattatgct N.D. 128                                   Codon                                                                         31203 Stop 1584 tgttcgtactaccattatgc N.D. 129                                   Codon                                                                         31204 Stop 1585 atgttcgtactaccattatg N.D. 130                                   Codon                                                                         31205 Stop 1586 tatgttcgtactaccattat N.D. 131                                   Codon                                                                         31206 Stop 1587 ttatgttcgtactaccatta N.D. 132                                   Codon                                                                         31207 Stop 1588 tttatgttcgtactaccatt N.D. 133                                   Codon                                                                         31208 Stop 1589 ttttatgttcgtactaccat N.D. 134                                   Codon                                                                         31209 3' UTR 1617 atgtggtaagtgaactgaat N.D. 135                                31052 3' UTR 1629 gattttcaaataatgtggta 80 136                                  31210 3' UTR 1645 aattaaataaaggattgatt N.D. 137                                31211 3' UTR 1647 aaaattaaataaaggattga N.D. 138                                31053 3' UTR 1662 tgacaggttggaaataaaat 78 139                                  31212 3' UTR 1669 cattctctgacaggttggaa N.D. 140                                31054 3' UTR 1685 ttttgatgcctaagaacatt 82 141                                  31213 3' UTR 1690 ttggattttgatgcctaaga N.D. 142                                31055 3' UTR 1702 cttacagctaccttggattt 82 143                                  31214 3' UTR 1725 tcattgttagctccagtatt N.D. 144                                31056 3' UTR 1738 ttacttctgttcttcattgt 83 145                                  31215 3' UTR 1759 ttaataatttgactaatcag N.D. 146                                31057 3' UTR 1787 gactgctgcataaagtaatc 82 147                                  31216 3' UTR 1800 taactatgtacctgactgct N.D. 148                                31058 3' UTR 1813 ttttgggttcacctaactat 86 149                                  31217 3' UTR 1853 aatgtgcatggaagaaatct N.D. 150                                31059 3' UTR 1878 atgttaattatacctcaata 78 151                                  31218 3' UTR 1899 gttagaaggaaacactttat N.D. 152                                31060 3' UTR 1914 agatttctacaactcgttag 23 153                                  31219 3' UTR 1919 tactcagatttctacaactc N.D. 154                                31061 3' UTR 1930 ttttgggtggttactcagat 86 155                                  31220 3' UTR 1974 ggcagaagggagtgttttgg N.D. 156                                31062 3' UTR 1996 gctgaaggactgtctgaaga 86 157                                  31063 3' UTR 2079 cctgtcgccccacacctccc 62 159                                  31221 3' UTR 2087 agacagaccctgtcgcccca N.D. 160                                31064 3' UTR 2102 ctgggagacagaacaagaca 81 161                                  31222 3' UTR 2110 acttcagcctgggagacaga N.D. 162                                31065 3' UTR 2130 gctcaatcttgactcactgc 16 163                                  31223 3' UTR 2144 gctggagtgcagtggctcaa N.D. 164                                31066 3' UTR 2160 tcgcgctgtcacccaggctg 14 165                                  31224 3' UTR 2168 atggagtctcgcgctgtcac N.D. 166                              __________________________________________________________________________

As shown in Table 1 SEQ ID NOs 10, 12, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 29, 30, 32, 34, 36, 38, 40, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 64, 66, 68, 71, 73, 74, 76, 78, 79, 81, 84, 86, 89, 91, 93, 95, 99, 101, 103, 105, 109, 112, 114, 116, 118, 121, 122, 125, 136, 139, 141, 143, 145, 147, 149, 151, 155, 157, 159 and 161 demonstrated at least 30% inhibition of MDMX expression in this experiment and are therefore preferred.

Example 16

Antisense Inhibition of MDMX Expression--Phosphorothioate Oligodeoxynucleotides

In accordance with the present invention, a second series of oligonucleotides were designed to target different regions of the human MDMX RNA, using published sequences (GenBank accession number AF007111, incorporated herein as SEQ ID NO: 1). The oligonucleotides are shown in Table 2. Target sites are indicated by nucleotide numbers, as given in the sequence source reference (Genbank accession no. AF007111), to which the oligonucleotide binds. All compounds in Table 2 are oligodeoxynucleotides with phosphorothioate backbones (internucleoside linkages) throughout. The compounds are analyzed for effect on MDMX mRNA levels by quantitative real-time PCR as described in other examples herein.

                  TABLE 2                                                          ______________________________________                                         Inhibition of MDMX mRNA levels by phosphorothioate                               oligodeoxynucleotides                                                                           TARGET              SEQ ID                                    ISIS# REGION SITE SEQUENCE NO.                                               ______________________________________                                         31067 5' UTR   58        cggcagctccatgagtcgct                                                                       9                                           31068 5' UTR 81 ctgcagtctgttggtaaaac 11                                        31069 5' UTR 87 aagaaactgcagtctgttgg 13                                        31070 Coding 123 tgagcagaggtggaaaatga 27                                       31071 Coding 145 agcactgtcagatgttgaac 28                                       31072 Coding 173 gattgatttgtccaggagag 31                                       31073 Coding 209 gcaaaatcttcaaaagcggc 33                                       31074 Coding 217 tgctgcatgcaaaatcttca 35                                       31075 Coding 253 gacctctttaacagtgaaca 37                                       31076 Coding 275 tgtactgacctaaatagtgc 39                                       31077 Coding 303 tgctgatcataaagttgctt 42                                       31078 Coding 323 aatataccatatgctgctcc 44                                       31079 Coding 360 tgacgtcccagtagttctcc 46                                       31080 Coding 388 agggcttgggttctttacgg 48                                       31081 Coding 407 ttcttagcatatcatagaga 50                                       31082 Coding 443 catctgtagtagcagtggct 52                                       31083 Coding 460 agcgagagtctgagcagcat 54                                       31084 Coding 482 ccatactgtgatcctgtgcg 56                                       31085 Coding 512 tttgcttcagttggtcttga 58                                       31086 Coding 525 ctttcctctgcactttgctt 60                                       31087 Coding 584 gctctgaggtaggcagtgtg 61                                       31088 Coding 623 caattaagtcttcatcttct 63                                       31089 Coding 672 tcctcaaatccaaggtccag 65                                       31090 Coding 686 cagctacatcccactcctca 67                                       31091 Coding 724 gttgcttctcaagtttccta 69                                       31092 Coding 753 tcagttgagccattacttct 70                                       31093 Coding 776 ccacatcctgatttgtctgt 72                                       31094 Coding 814 ccacaagtcatctgtagtat 75                                       31095 Coding 859 ttttattccaacacctaact 77                                       31096 Coding 901 ccctacttcttcacttgttt 80                                       31097 Coding 986 ctacatcggtatcatcactt 82                                       31098 Coding 992 taacctctacatcggtatca 83                                       31099 Coding 1018 agtacactgccactcatcct 85                                      31100 Coding 1075 caaggcccaacaacgaaaac 87                                      31101 Coding 1080 ttcctcaaggcccaacaacg 88                                      31102 Coding 1103 ttgaacaatctgaataccaa 90                                      31103 Coding 1117 agaatgggttaactttgaac 92                                      31104 Coding 1152 ttttcaggtatggcagtgat 94                                      31105 Coding 1168 atttccttcattttcctttt 96                                      31106 Coding 1180 atcagggacatcatttcctt 98                                      31107 Coding 1195 aatggttcttcgacaatcag 100                                     31108 Coding 1218 ttaggtctaacgacaggagc 102                                   ______________________________________                                    

Example 17

Western Blot Analysis of MDMX Protein Levels

Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to MDMX is used, with a radiolabelled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

    __________________________________________________________________________     #             SEQUENCE LISTING                                                    - -  - - <160> NUMBER OF SEQ ID NOS: 166                                       - - <210> SEQ ID NO 1                                                         <211> LENGTH: 2192                                                             <212> TYPE: DNA                                                                <213> ORGANISM: Homo sapiens                                                   <220> FEATURE:                                                                 <221> NAME/KEY: CDS                                                            <222> LOCATION: (117)..(1589)                                                   - - <400> SEQUENCE: 1                                                          - - cggcacgagc taggatctgt gactgccacc cctcccccca cccgggctcg gc -             #gggggagc     60                                                                  - - gactcatgga gctgccgtaa gttttaccaa cagactgcag tttcttcact ac - #caaa             116                                                                        - - atg aca tca ttt tcc acc tct gct cag tgt tc - #a aca tct gac agt gct           164                                                                        Met Thr Ser Phe Ser Thr Ser Ala Gln Cys Se - #r Thr Ser Asp Ser Ala              1               5 - #                 10 - #                 15               - - tgc agg atc tct cct gga caa atc aat cag gt - #a cga cca aaa ctg ccg           212                                                                        Cys Arg Ile Ser Pro Gly Gln Ile Asn Gln Va - #l Arg Pro Lys Leu Pro                         20     - #             25     - #             30                   - - ctt ttg aag att ttg cat gca gca ggt gcg ca - #a ggt gaa atg ttc act           260                                                                        Leu Leu Lys Ile Leu His Ala Ala Gly Ala Gl - #n Gly Glu Met Phe Thr                     35         - #         40         - #         45                       - - gtt aaa gag gtc atg cac tat tta ggt cag ta - #c ata atg gtg aag caa           308                                                                        Val Lys Glu Val Met His Tyr Leu Gly Gln Ty - #r Ile Met Val Lys Gln                 50             - #     55             - #     60                           - - ctt tat gat cag cag gag cag cat atg gta ta - #t tgt ggt gga gat ctt           356                                                                        Leu Tyr Asp Gln Gln Glu Gln His Met Val Ty - #r Cys Gly Gly Asp Leu             65                 - # 70                 - # 75                 - # 80        - - ttg gga gaa cta ctg gga cgt cag agc ttc tc - #c gta aag aac cca agc           404                                                                        Leu Gly Glu Leu Leu Gly Arg Gln Ser Phe Se - #r Val Lys Asn Pro Ser                             85 - #                 90 - #                 95               - - cct ctc tat gat atg cta aga aag aat ctt gt - #c act tta gcc act gct           452                                                                        Pro Leu Tyr Asp Met Leu Arg Lys Asn Leu Va - #l Thr Leu Ala Thr Ala                        100      - #           105      - #           110                   - - act aca gat gct gct cag act ctc gct ctc gc - #a cag gat cac agt atg           500                                                                        Thr Thr Asp Ala Ala Gln Thr Leu Ala Leu Al - #a Gln Asp His Ser Met                    115          - #       120          - #       125                       - - gat att cca agt caa gac caa ctg aag caa ag - #t gca gag gaa agt tcc           548                                                                        Asp Ile Pro Ser Gln Asp Gln Leu Lys Gln Se - #r Ala Glu Glu Ser Ser                130              - #   135              - #   140                           - - act tcc aga aaa aga act aca gaa gac gat at - #c ccc aca ctg cct acc           596                                                                        Thr Ser Arg Lys Arg Thr Thr Glu Asp Asp Il - #e Pro Thr Leu Pro Thr            145                 1 - #50                 1 - #55                 1 -       #60                                                                               - - tca gag cat aaa tgc ata cat tct aga gaa ga - #t gaa gac tta att         gaa      644                                                                     Ser Glu His Lys Cys Ile His Ser Arg Glu As - #p Glu Asp Leu Ile Glu                           165  - #               170  - #               175               - - aat tta gcc caa gat gaa aca tct agg ctg ga - #c ctt gga ttt gag gag           692                                                                        Asn Leu Ala Gln Asp Glu Thr Ser Arg Leu As - #p Leu Gly Phe Glu Glu                        180      - #           185      - #           190                   - - tgg gat gta gct ggc ctg cct tgg tgg ttt tt - #a gga aac ttg aga agc           740                                                                        Trp Asp Val Ala Gly Leu Pro Trp Trp Phe Le - #u Gly Asn Leu Arg Ser                    195          - #       200          - #       205                       - - aac tat aca cct aga agt aat ggc tca act ga - #t tta cag aca aat cag           788                                                                        Asn Tyr Thr Pro Arg Ser Asn Gly Ser Thr As - #p Leu Gln Thr Asn Gln                210              - #   215              - #   220                           - - gat gtg ggt act gcc att gtt tca gat act ac - #a gat gac ttg tgg ttt           836                                                                        Asp Val Gly Thr Ala Ile Val Ser Asp Thr Th - #r Asp Asp Leu Trp Phe            225                 2 - #30                 2 - #35                 2 -       #40                                                                               - - ttg aat gag tca gta tca gag cag tta ggt gt - #t gga ata aaa gtt         gaa      884                                                                     Leu Asn Glu Ser Val Ser Glu Gln Leu Gly Va - #l Gly Ile Lys Val Glu                           245  - #               250  - #               255               - - gct gct gat act gaa caa aca agt gaa gaa gt - #a ggg aaa gta agt gac           932                                                                        Ala Ala Asp Thr Glu Gln Thr Ser Glu Glu Va - #l Gly Lys Val Ser Asp                        260      - #           265      - #           270                   - - aaa aag gtg att gaa gtg gga aaa aat gat ga - #c ctg gag gac tct aag           980                                                                        Lys Lys Val Ile Glu Val Gly Lys Asn Asp As - #p Leu Glu Asp Ser Lys                    275          - #       280          - #       285                       - - tcc tta agt gat gat acc gat gta gag gtt ac - #c tct gag gat gag tgg          1028                                                                        Ser Leu Ser Asp Asp Thr Asp Val Glu Val Th - #r Ser Glu Asp Glu Trp                290              - #   295              - #   300                           - - cag tgt act gaa tgc aag aaa ttt aac tct cc - #a agc aag agg tac tgt          1076                                                                        Gln Cys Thr Glu Cys Lys Lys Phe Asn Ser Pr - #o Ser Lys Arg Tyr Cys            305                 3 - #10                 3 - #15                 3 -       #20                                                                               - - ttt cgt tgt tgg gcc ttg agg aag gat tgg ta - #t tca gat tgt tca         aag     1124                                                                     Phe Arg Cys Trp Ala Leu Arg Lys Asp Trp Ty - #r Ser Asp Cys Ser Lys                           325  - #               330  - #               335               - - tta acc cat tct ctc tcc acg tct gat atc ac - #t gcc ata cct gaa aag          1172                                                                        Leu Thr His Ser Leu Ser Thr Ser Asp Ile Th - #r Ala Ile Pro Glu Lys                        340      - #           345      - #           350                   - - gaa aat gaa gga aat gat gtc cct gat tgt cg - #a aga acc att tcg gct          1220                                                                        Glu Asn Glu Gly Asn Asp Val Pro Asp Cys Ar - #g Arg Thr Ile Ser Ala                    355          - #       360          - #       365                       - - cct gtc gtt aga cct aaa gat gcg tat ata aa - #g aaa gaa aac tcc aaa          1268                                                                        Pro Val Val Arg Pro Lys Asp Ala Tyr Ile Ly - #s Lys Glu Asn Ser Lys                370              - #   375              - #   380                           - - ctt ttt gat ccc tgc aac tca gtg gaa ttc tt - #g gat ttg gct cac agt          1316                                                                        Leu Phe Asp Pro Cys Asn Ser Val Glu Phe Le - #u Asp Leu Ala His Ser            385                 3 - #90                 3 - #95                 4 -       #00                                                                               - - tct gaa agc caa gag acc atc tca agc atg gg - #a gaa cag tta gat         aac     1364                                                                     Ser Glu Ser Gln Glu Thr Ile Ser Ser Met Gl - #y Glu Gln Leu Asp Asn                           405  - #               410  - #               415               - - ctt tct gaa cag aga aca gat aca gaa aac at - #g gag gat tgc cag aat          1412                                                                        Leu Ser Glu Gln Arg Thr Asp Thr Glu Asn Me - #t Glu Asp Cys Gln Asn                        420      - #           425      - #           430                   - - ctc ttg aag cca tgt agc tta tgt gag aaa ag - #a cca cga gac ggg aac          1460                                                                        Leu Leu Lys Pro Cys Ser Leu Cys Glu Lys Ar - #g Pro Arg Asp Gly Asn                    435          - #       440          - #       445                       - - att att cat gga agg acg ggc cat ctt gtc ac - #t tgt ttt cac tgt gcc          1508                                                                        Ile Ile His Gly Arg Thr Gly His Leu Val Th - #r Cys Phe His Cys Ala                450              - #   455              - #   460                           - - aga aga cta aag aag gct ggg gct tca tgc cc - #t att tgc aag aaa gag          1556                                                                        Arg Arg Leu Lys Lys Ala Gly Ala Ser Cys Pr - #o Ile Cys Lys Lys Glu            465                 4 - #70                 4 - #75                 4 -       #80                                                                               - - att cag ctg gtt att aag gtt ttt ata gca ta - #a tggtagtacg              aacataaaaa   1609                                                                Ile Gln Leu Val Ile Lys Val Phe Ile Ala                                                        485  - #               490                                      - - tgcatttatt cagttcactt accacattat ttgaaaatca atcctttatt ta -             #attttatt   1669                                                                  - - tccaacctgt cagagaatgt tcttaggcat caaaatccaa ggtagctgta ag -             #aaaaatac   1729                                                                  - - tggagctaac aatgaagaac agaagtaatc tgattagtca aattattaag tg -             #ccatggat   1789                                                                  - - tactttatgc agcagtcagg tacatagtta ggtgaaccca aaagaaaaac tc -             #ttgaaaac   1849                                                                  - - aagagatttc ttccatgcac atttacaata ttgaggtata attaacatga ta -             #aagtgttt   1909                                                                  - - ccttctaacg agttgtagaa atctgagtaa ccacccaaaa aagcaataga at -             #gtttgtgt   1969                                                                  - - caccccaaaa cactcccttc tgcccctctt cagacagtcc ttcagctatt tc -             #atggctct   2029                                                                  - - caccctagtt tttttttttt tttgcacttt tttttttccg ggggtatagg gg -             #aggtgtgg   2089                                                                  - - ggcgacaggg tctgtcttgt tctgtctccc aggctgaagt gcagtgagtc aa -             #gattgagc   2149                                                                  - - cactgcactc cagcctgggt gacagcgcga gactccatct cag    - #                      219 - #2                                                                     - -  - - <210> SEQ ID NO 2                                                    <211> LENGTH: 24                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: PCR Primer                                             - - <400> SEQUENCE: 2                                                          - - tgccgtaagt tttaccaaca gact          - #                  - #                     24                                                                       - -  - - <210> SEQ ID NO 3                                                    <211> LENGTH: 22                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: PCR Primer                                             - - <400> SEQUENCE: 3                                                          - - ggcagttttg gtcgtacctg at           - #                  - #                      22                                                                       - -  - - <210> SEQ ID NO 4                                                    <211> LENGTH: 32                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: PCR Probe                                              - - <400> SEQUENCE: 4                                                          - - tcattttcca cctctgctca gtgttcaaca tc       - #                  - #                      32                                                                - -  - - <210> SEQ ID NO 5                                                    <211> LENGTH: 19                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: PCR Primer                                             - - <400> SEQUENCE: 5                                                          - - gaaggtgaag gtcggagtc             - #                  - #                       - # 19                                                                    - -  - - <210> SEQ ID NO 6                                                    <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: PCR Primer                                             - - <400> SEQUENCE: 6                                                          - - gaagatggtg atgggatttc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 7                                                    <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: PCR Probe                                              - - <400> SEQUENCE: 7                                                          - - caagcttccc gttctcagcc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 8                                                    <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 8                                                          - - cagctccatg agtcgctccc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 9                                                    <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 9                                                          - - cggcagctcc atgagtcgct            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 10                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 10                                                         - - gcagtctgtt ggtaaaactt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 11                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 11                                                         - - ctgcagtctg ttggtaaaac            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 12                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 12                                                         - - aaactgcagt ctgttggtaa            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 13                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 13                                                         - - aagaaactgc agtctgttgg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 14                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 14                                                         - - ttttggtagt gaagaaactg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 15                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 15                                                         - - attttggtag tgaagaaact            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 16                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 16                                                         - - cattttggta gtgaagaaac            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 17                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 17                                                         - - tcattttggt agtgaagaaa            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 18                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 18                                                         - - gtcattttgg tagtgaagaa            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 19                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 19                                                         - - tgtcattttg gtagtgaaga            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 20                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 20                                                         - - atgtcatttt ggtagtgaag            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 21                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 21                                                         - - gatgtcattt tggtagtgaa            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 22                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 22                                                         - - tgatgtcatt ttggtagtga            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 23                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 23                                                         - - atgatgtcat tttggtagtg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 24                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 24                                                         - - aatgatgtca ttttggtagt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 25                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 25                                                         - - agaggtggaa aatgatgtca            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 26                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 26                                                         - - cagaggtgga aaatgatgtc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 27                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 27                                                         - - tgagcagagg tggaaaatga            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 28                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 28                                                         - - agcactgtca gatgttgaac            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 29                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 29                                                         - - caagcactgt cagatgttga            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 30                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 30                                                         - - gatcctgcaa gcactgtcag            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 31                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 31                                                         - - gattgatttg tccaggagag            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 32                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 32                                                         - - agttttggtc gtacctgatt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 33                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 33                                                         - - gcaaaatctt caaaagcggc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 34                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 34                                                         - - catgcaaaat cttcaaaagc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 35                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 35                                                         - - tgctgcatgc aaaatcttca            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 36                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 36                                                         - - tctttaacag tgaacatttc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 37                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 37                                                         - - gacctcttta acagtgaaca            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 38                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 38                                                         - - aatagtgcat gacctcttta            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 39                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 39                                                         - - tgtactgacc taaatagtgc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 40                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 40                                                         - - tcaccattat gtactgacct            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 41                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 41                                                         - - atcataaagt tgcttcacca            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 42                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 42                                                         - - tgctgatcat aaagttgctt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 43                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 43                                                         - - ataccatatg ctgctcctgc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 44                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 44                                                         - - aatataccat atgctgctcc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 45                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 45                                                         - - cagtagttct cccaaaagat            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 46                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 46                                                         - - tgacgtccca gtagttctcc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 47                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 47                                                         - - agctctgacg tcccagtagt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 48                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 48                                                         - - agggcttggg ttctttacgg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 49                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 49                                                         - - agcatatcat agagagggct            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 50                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 50                                                         - - ttcttagcat atcatagaga            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 51                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 51                                                         - - gtggctaaag tgacaagatt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 52                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 52                                                         - - catctgtagt agcagtggct            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 53                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 53                                                         - - ctgagcagca tctgtagtag            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 54                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 54                                                         - - agcgagagtc tgagcagcat            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 55                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 55                                                         - - tgtgatcctg tgcgagagcg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 56                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 56                                                         - - ccatactgtg atcctgtgcg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 57                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 57                                                         - - cagttggtct tgacttggaa            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 58                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 58                                                         - - tttgcttcag ttggtcttga            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 59                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 59                                                         - - tctgcacttt gcttcagttg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 60                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 60                                                         - - ctttcctctg cactttgctt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 61                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 61                                                         - - gctctgaggt aggcagtgtg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 62                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 62                                                         - - atgcatttat gctctgaggt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 63                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 63                                                         - - caattaagtc ttcatcttct            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 64                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 64                                                         - - aaggtccagc ctagatgttt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 65                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 65                                                         - - tcctcaaatc caaggtccag            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 66                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 66                                                         - - acatcccact cctcaaatcc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 67                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 67                                                         - - cagctacatc ccactcctca            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 68                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 68                                                         - - aaggcaggcc agctacatcc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 69                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 69                                                         - - gttgcttctc aagtttccta            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 70                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 70                                                         - - tcagttgagc cattacttct            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 71                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 71                                                         - - gtctgtaaat cagttgagcc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 72                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 72                                                         - - ccacatcctg atttgtctgt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 73                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 73                                                         - - caatggcagt acccacatcc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 74                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 74                                                         - - atctgtagta tctgaaacaa            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 75                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 75                                                         - - ccacaagtca tctgtagtat            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 76                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 76                                                         - - caacacctaa ctgctctgat            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 77                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 77                                                         - - ttttattcca acacctaact            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 78                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 78                                                         - - tatcagcagc ttcaactttt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 79                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 79                                                         - - cacttgtttg ttcagtatca            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 80                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 80                                                         - - ccctacttct tcacttgttt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 81                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 81                                                         - - agtcctccag gtcatcattt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 82                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 82                                                         - - ctacatcggt atcatcactt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 83                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 83                                                         - - taacctctac atcggtatca            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 84                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 84                                                         - - gaggtaacct ctacatcggt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 85                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 85                                                         - - agtacactgc cactcatcct            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 86                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 86                                                         - - taaatttctt gcattcagta            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 87                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 87                                                         - - caaggcccaa caacgaaaac            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 88                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 88                                                         - - ttcctcaagg cccaacaacg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 89                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 89                                                         - - atctgaatac caatccttcc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 90                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 90                                                         - - ttgaacaatc tgaataccaa            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 91                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 91                                                         - - gttaactttg aacaatctga            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 92                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 92                                                         - - agaatgggtt aactttgaac            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 93                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 93                                                         - - tatcagacgt ggagagagaa            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 94                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 94                                                         - - ttttcaggta tggcagtgat            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 95                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 95                                                         - - cattttcctt ttcaggtatg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 96                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 96                                                         - - atttccttca ttttcctttt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 97                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 97                                                         - - acatcatttc cttcattttc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 98                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 98                                                         - - atcagggaca tcatttcctt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 99                                                   <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 99                                                         - - tcttcgacaa tcagggacat            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 100                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 100                                                        - - aatggttctt cgacaatcag            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 101                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 101                                                        - - aacgacagga gccgaaatgg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 102                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 102                                                        - - ttaggtctaa cgacaggagc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 103                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 103                                                        - - tatatacgca tctttaggtc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 104                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 104                                                        - - ttctttatat acgcatcttt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 105                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 105                                                        - - caagaattcc actgagttgc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 106                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 106                                                        - - ctgtgagcca aatccaagaa            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 107                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 107                                                        - - ttggctttca gaactgtgag            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 108                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 108                                                        - - atggtctctt ggctttcaga            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 109                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 109                                                        - - actgttctcc catgcttgag            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 110                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 110                                                        - - aggttatcta actgttctcc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 111                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 111                                                        - - tctgttcaga aaggttatct            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 112                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 112                                                        - - ttctgtatct gttctctgtt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 113                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 113                                                        - - ctccatgttt tctgtatctg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 114                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 114                                                        - - caatcctcca tgttttctgt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 115                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 115                                                        - - catggcttca agagattctg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 116                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 116                                                        - - tcgtggtctt ttctcacata            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 117                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 117                                                        - - cccgtctcgt ggtcttttct            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 118                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 118                                                        - - aataatgttc ccgtctcgtg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 119                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 119                                                        - - ccttccatga ataatgttcc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 120                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 120                                                        - - gacaagatgg cccgtccttc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 121                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 121                                                        - - agtgaaaaca agtgacaaga            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 122                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 122                                                        - - ttctggcaca gtgaaaacaa            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 123                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 123                                                        - - ttctttagtc ttctggcaca            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 124                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 124                                                        - - tctttcttgc aaatagggca            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 125                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 125                                                        - - ttaataacca gctgaatctc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 126                                                  <211> LENGTH:                                                                  <212> TYPE:                                                                    <213> ORGANISM:                                                                 - - <400> SEQUENCE: 126                                                        - -       This Sequence is intentionally skipp - #ed                           - -  - - <210> SEQ ID NO 127                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 127                                                        - - ttcgtactac cattatgcta            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 128                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 128                                                        - - gttcgtacta ccattatgct            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 129                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 129                                                        - - tgttcgtact accattatgc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 130                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 130                                                        - - atgttcgtac taccattatg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 131                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 131                                                        - - tatgttcgta ctaccattat            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 132                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 132                                                        - - ttatgttcgt actaccatta            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 133                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 133                                                        - - tttatgttcg tactaccatt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 134                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 134                                                        - - ttttatgttc gtactaccat            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 135                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 135                                                        - - atgtggtaag tgaactgaat            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 136                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 136                                                        - - gattttcaaa taatgtggta            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 137                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 137                                                        - - aattaaataa aggattgatt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 138                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 138                                                        - - aaaattaaat aaaggattga            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 139                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 139                                                        - - tgacaggttg gaaataaaat            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 140                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 140                                                        - - cattctctga caggttggaa            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 141                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 141                                                        - - ttttgatgcc taagaacatt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 142                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 142                                                        - - ttggattttg atgcctaaga            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 143                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 143                                                        - - cttacagcta ccttggattt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 144                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 144                                                        - - tcattgttag ctccagtatt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 145                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 145                                                        - - ttacttctgt tcttcattgt            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 146                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 146                                                        - - ttaataattt gactaatcag            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 147                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 147                                                        - - gactgctgca taaagtaatc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 148                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 148                                                        - - taactatgta cctgactgct            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 149                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 149                                                        - - ttttgggttc acctaactat            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 150                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 150                                                        - - aatgtgcatg gaagaaatct            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 151                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 151                                                        - - atgttaatta tacctcaata            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 152                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 152                                                        - - gttagaagga aacactttat            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 153                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 153                                                        - - agatttctac aactcgttag            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 154                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 154                                                        - - tactcagatt tctacaactc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 155                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 155                                                        - - ttttgggtgg ttactcagat            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 156                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 156                                                        - - ggcagaaggg agtgttttgg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 157                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 157                                                        - - gctgaaggac tgtctgaaga            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 158                                                  <211> LENGTH:                                                                  <212> TYPE:                                                                    <213> ORGANISM:                                                                 - - <400> SEQUENCE: 158                                                        - -       This Sequence is intentionally skipp - #ed                           - -  - - <210> SEQ ID NO 159                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 159                                                        - - cctgtcgccc cacacctccc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 160                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 160                                                        - - agacagaccc tgtcgcccca            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 161                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 161                                                        - - ctgggagaca gaacaagaca            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 162                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 162                                                        - - acttcagcct gggagacaga            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 163                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 163                                                        - - gctcaatctt gactcactgc            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 164                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 164                                                        - - gctggagtgc agtggctcaa            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 165                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 165                                                        - - tcgcgctgtc acccaggctg            - #                  - #                       - # 20                                                                    - -  - - <210> SEQ ID NO 166                                                  <211> LENGTH: 20                                                               <212> TYPE: DNA                                                                <213> ORGANISM: Artificial Sequence                                            <220> FEATURE:                                                                 <223> OTHER INFORMATION: Antisense Oligonucleotide                              - - <400> SEQUENCE: 166                                                        - - atggagtctc gcgctgtcac            - #                  - #                       - # 20                                                                  __________________________________________________________________________ 

What is claimed is:
 1. An antisense compound 8 to 30 nucleobases in length targeted to a nucleic acid molecule encoding human MDMX or wherein said antisense compound specifically hybridizes with and inhibits the expression of human MDMX.
 2. The antisense compound of claim 1 which is an antisense oligonucleotide.
 3. The antisense compound of claim 2 wherein the antisense oligonucleotide has a sequence comprising SEQ ID NO: 10, 12, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 29, 30, 32, 34, 36, 38, 40, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 64, 66, 68, 71, 73, 74, 76, 78, 79, 81, 84, 86, 89, 91, 93, 95, 99, 101, 103, 105, 109, 112, 114, 116, 118, 121, 122, 125, 136, 139, 141, 143, 145, 147, 149, 151, 155, 157, 159 or
 161. 4. The antisense compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified internucleoside linkage.
 5. The antisense compound of claim 4 wherein the modified internucleoside linkage is a phosphorothioate linkage.
 6. The antisense compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified sugar moiety.
 7. The antisense compound of claim 6 wherein the modified sugar moiety is a 2'-O-methoxyethyl sugar moiety.
 8. The antisense compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified nucleobase.
 9. The antisense compound of claim 8 wherein the modified nucleobase is a 5-methylcytosine.
 10. The antisense compound of claim 1 wherein the antisense oligonucleotide is a chimeric oligonucleotide.
 11. A composition comprising the antisense compound of claim 1 and a carrier or diluent.
 12. The composition of claim 11 further comprising a colloidal dispersion system.
 13. The composition of claim 11 wherein the antisense compound is an antisense oligonucleotide.
 14. A method of inhibiting the expression of human MDMX in human cells or tissues in vitro comprising contacting said cells or tissues with the antisense compound of claim 1 so that expression of human MDMX is inhibited. 